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Perhydrolase providing improved specific activity

a technology of peroxycarboxylic acid and specific activity, which is applied in the direction of enzyme stabilisation, depsipeptides, and vector-based foreign material introduction, can solve the problem of reducing the amount of enzyme used to achieve the desired concentration of peroxycarboxylic acid, and achieve the effect of increasing specific activity

Inactive Publication Date: 2011-09-29
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a nucleic acid molecule that encodes a perhydrolytic enzyme with increased activity compared to a specific enzyme found in Thermotoga maritima. The mutated nucleic acid molecule was created by modifying the amino acid sequence of the original enzyme. The mutated nucleic acid molecule can be used to create a library of variant enzymes with perhydrolytic activity. The patent also describes a method for transforming a cell using the mutated nucleic acid molecule. The mutated nucleic acid molecule can be used to produce peroxycarboxylic acid, which can be used for disinfecting, sanitizing, bleaching, or any other applications where its unique properties are useful. The patent also provides a composition containing the mutated nucleic acid molecule and a set of reaction components for creating a peroxycarboxylic acid.

Problems solved by technology

However, there remains an ongoing need to identify additional variants having even higher perhydrolytic specific activity as a relative increase in specific activity reduces the amount of enzyme used to achieve a desired peroxycarboxylic acid concentration.

Method used

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  • Perhydrolase providing improved specific activity

Examples

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Effect test

example 1

Construction of a Random Mutagenesis Library of Thermotoga maritima Acetyl Xylan Esterase C277S Variant

[0216]The coding sequence of a Thermotoga maritima acetyl xylan esterase (GENBANK® accession # NP—227893.1) was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.), and cloned into pUC19 between Pst1 and Xba1 to create the plasmid known as pSW202 (U.S. Patent Application Publication No. 2008-0176299). The codon-optimized sequence is provided as SEQ ID NO: 1 encoding the wild-type Thermotoga maritima acetyl xylan esterase provided as SEQ ID NO: 2.

[0217]A codon change was made in the gene using primer pairs identified as SEQ ID NO: 3 and SEQ ID NO: 4, and the QUIKCHANGE® site-directed mutagenesis kit (Stratagene, La Jolla, Calif.), according to the manufacturer's instructions, resulting in the amino acid change C2775 (SEQ ID NO: 5), to create the plasmid known as pSW202 / C277S (SEQ ID NO: 6). Plasmid pSW202 / C277S served as a template for error-pr...

example 2

Screening of Thermotoga maritima Error-prone PCR Library for Increased Enzyme Activity

[0218]Colonies were picked (automated) and placed into 96-well “master plates” containing 0.1 mL LB media supplemented with 0.1 mg ampicillin / mL and grown 16-18 h at 37° C. and 80% humidity. From each well of the master plates, 0.003 mL of culture was transferred to 96-well “induction plates” containing 0.3 mL LB media supplemented with 0.1 mg ampicillin / mL and 0.5 mM IPTG, which were incubated for 16-18 h with shaking at 37° C. and 80% humidity. Separately, 0.1 mL of 50% glycerol was added to each well of the master plates, which were stored at −80° C. as stocks. From each well of the induction plates, 0.01 mL of culture was transferred to 96-well “lysis plates” containing 0.09 mL of 56 mg / mL CELLYTIC™ Express (Sigma Aldrich, St. Louis, Mo.), which were incubated for 30 minutes at 30° C. From each well of the lysis plates, 0.01 mL of material was transferred to 96-well “assay plates” containing 0....

example 3

Production of Variant Thermotoga maritima Perhydrolases

[0219]Variant strains identified in the screen (KLP18 / pSW202 / L8R / L125Q / Q176L / V183D / F247I / C277S / P292L; KLP18 / pSW202 / A206E / C277S; KLP18 / pSW202 / S35R / H50R / C277S; KLP18 / pSW202 / K77E / A266E / C277S; KLP18 / pSW202 / F27Y / I149V / A266V / C277S / I295T / N302S; KLP18 / pSW202 / G119S / L207P / C277S; KLP18 / pSW202 / L195Q / C277S; KLP18 / pSW202 / K52E / C277S / Y298F; KLP18 / pSW202 / L125Q / T249S / C277S / A311V; KLP18 / pSW202 / F38S / V197I / N275T / C277S; KLP18 / pSW202 / K77I / K126N / C277S / K293R; and KLP18 / pSW202 / Y110F / C277S) were grown in LB media at 37° C. with shaking up to OD600nm=0.4-0.5, at which time IPTG was added to a final concentration of 1 mM, and incubation continued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of the perhydrolase enzyme at 20-40% of total soluble protein.

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Abstract

An acetyl xylan esterase variant having perhydrolytic activity is provided for producing peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen. More specifically, a Thermotoga maritima acetyl xylan esterase gene was modified using error-prone PCR and site-directed mutagenesis to create an enzyme catalyst characterized by an increase in specific activity. The variant acetyl xylan esterase may be used to produce peroxycarboxylic acids suitable for use in a variety of applications such as cleaning, disinfecting, sanitizing, bleaching, wood pulp processing, and paper pulp processing applications.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims benefit of U.S. Provisional Patent Application No. 61 / 318,032, filed Mar. 26, 2010, which is incorporated by reference herein in its entirety.TECHNICAL FIELD[0002]The invention relates to the field of peroxycarboxylic acid biosynthesis and enzyme catalysis. More specifically, an enzyme catalyst comprising a variant enzyme having perhydrolytic activity is provided having an increase in specific activity. Methods of using the present enzyme catalyst to produce peroxycarboxylic acids are also provided.BACKGROUND[0003]Peroxycarboxylic acid compositions can be effective antimicrobial agents. Methods of using peroxycarboxylic acids to clean, disinfect, and / or sanitize hard surfaces, textiles, meat products, living plant tissues, and medical devices against undesirable microbial growth have been described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. Patent Application Publication No. 200...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/18C12N15/63C12N1/00C12P7/24C07H21/04A61L2/16A61L9/00D06L3/02D21C9/10
CPCC11D3/38636C12P7/54C12P7/40C12N9/18
Inventor DICOSIMO, ROBERTGAVAGAN, JOHN EDWARDPAYNE, MARK SCOTT
Owner EI DU PONT DE NEMOURS & CO
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